Zinc(ii)-Selective Ratiometric Fluorescent Sensors Based on Inhibition of Excited-State Intramolecular Proton Transfer Maged M. Henary, Yonggang Wu, and ChristophJ. Fahrni* [a] Introduction The development of cation-selective fluorescence sensors has been an important goal of coordination chemistry for decades. [1] Fluorescence-based probes provide high sensitivi- ty and are therefore particularly well suited for the visuali- zation of metal cations in biological environments. [2] The majority of fluorescence sensors function as cation-respon- sive optical switches that translate the binding event into an increase or decrease of the emission intensity. [3] Due to the linear relationship between intensity and cation concentra- tion, quantitative measurements are, in principle, possible; however, the emission intensity depends also on the sensor concentration, which is typically not known with sufficient accuracy in biological applications. Because the sensor enters the cell through passive diffusion across the plasma membrane, the intracellular concentration may vary as a function of incubation time, temperature, membrane perme- ability, and variations in cell size. Ratiometric probes that exhibit a spectral shift upon bind- ing of the cation offer an elegant solution to this problem. The ratio of the emission intensities at two excitation or emission wavelengths varies as a function of the cation con- centration, but is independent of the probe concentration, pathlength, or spectral sensitivity of the instrument. [4] This approach was originally developed for the visualization of calcium-ion fluxes in live cells, [5] and a number of ratiomet- ric sensors are now commercially available. [6] More recently, studies on various eukaryotic cell lines indicate the presence of intracellular compartments containing weakly bound, labile Zn II (presumably up to millimolar concentrations). To study the dynamics of compartmentalized Zn II in live cells quantitatively, a cell-permeable, emission ratiometric sensor would be very beneficial. Most of the currently available cell-permeable Zn II probes function only as fluorescence switches and are not suitable for ratiometric measure- ments. [17±22] Furthermore, due to the high cost of laser equip- ment, modern 3D-imaging tools, such as confocal and multi- photon microscopy, depend on a single-excitation light source. To take advantage of these new imaging techniques, the sensor must provide a spectral shift of the peak emis- sion; however, with the exception of IndoZin, [23] currently available cell-permeable ratiometric Zn II sensors require dual excitation and are therefore not suitable for these ap- plications. [6,23,24,52] To develop a Zn II -selective emission ratiometric probe, we recently explored the photophysics of cation-induced inhibi- tion of excited-state intramolecular proton transfer (ESIPT). [25] Benzimidazole derivatives (T A ) containing an intramolecular hydrogen bond undergo ESIPT and yield a highly Stokes× shifted emission from the proton-transfer tau- tomer T * B (Scheme 1). [26±30] If the acidity of the proton at- [a] Dr. M. M. Henary, Y. Wu, Prof. Dr. C. J. Fahrni School of Chemistry and Biochemistry Georgia Institute of Technology 770 State Street, Atlanta, GA 30332 (U.S.A.) Fax: (+ 1)404-894-2295 E-mail: fahrni@chemistry.gatech.edu Abstract: To develop a zinc(ii )-selec- tive emission ratiometric probe suitable for biological applications, we explored the cation-induced inhibition of excit- ed-state intramolecular proton transfer (ESIPT) with a series of 2-(2’-benzene- sulfonamidophenyl)benzimidazole de- rivatives. In the absence of Zn II at neu- tral pH, the fluorophores undergo ESIPT to yield a highly Stokes× shifted emission from the proton-transfer tau- tomer. Coordination of Zn II inhibits the ESIPT process and yields a signifi- cant hypsochromic shift of the fluores- cence emission maximum. Whereas the paramagnetic metal cations Cu II , Fe II , Ni II , Co II , and Mn II result in fluores- cence quenching, the emission response is not altered by millimolar concentra- tions of Ca II or Mg II , rendering the sen- sors selective for Zn II among all biolog- ically important metal cations. Due to the modular architecture of the fluoro- phore, the Zn II binding affinity can be readily tuned by implementing simple structural modifications. The synthe- sized probes are suitable to gauge free Zn II concentrations in the micromolar to picomolar range under physiological conditions. Keywords: fluorescent probes ¥ ligand design ¥ proton transfer ¥ sensors ¥ zinc Chem. Eur. J. 2004, 10, 3015±3025 DOI: 10.1002/chem.200305299 ¹ 2004 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 3015 FULL PAPER